DESCRIPTION (adapted from the abstract): Helicobacter pylori causes gastritis and is strongly associated with peptic ulcers and gastric carcinoma. Central to the pathogenesis of H. pylori is the copious urease produced. Twenty-three independent Lambda ZapII clones with qualitatively enhanced urease activity have been isolated from E. coli cotransformed with pHP808 (ureABIEFGH). Two of these clones, pUEF201 and pUEF728, contain the nickel transporter nixA and the glutamine synthetase glnA, respectively, and are currently being characterized. We propose to analyze the remaining 21 Lambda ZapII clones in E. coli (pHP808) for quantitative production of urease activity. Confirmed urease-enhancing clones will be assayed for nickel transport and nickel ion incorporation into apo-urease, given the requirement for nickel for urease activity. Clones of interest will be subcloned, sequenced and the gene(s) disrupted by an antibiotic cassette. These disruptions will be transformed into H. pylori and confirmed allelic exchange mutants will be assayed for urease activity, nickel transport and nickel ion incorporation into apo-urease. As three of these Lambda ZapII clones have already been preliminary shown to have quantitatively enhanced urease activity in E. coli (pHP808), we hypothesize that disruption of the genes responsible will result in reduction of urease activity once inactivated in H. pylori. These experiments should reveal more details about how apo-urease is converted into the active enzyme, how nickel ions are utilized by H. pylori for the production of catalytically active urease, and how urease contributes to the overall pathogenicity of H. pylori infection. These results may lead to novel therapeutic and/or vaccine strategies to treat H. pylori infection.